Radiant Genomics proposes to develop an integrated enzyme discovery service, the Enzyme Variant Engine (EVE), built upon the largest cloned metagenomic sequence collection reported to date. The goal is to combine a publiclyaccessible search engine, richlyannotated sequence database, arrayed sample library, and LIMS automation platform to deliver novel enzyme variants to endusers for lower cost, in less time, and from a greater pool of biodiversity than alternative options, such as DNA synthesis. Importantly, this service overcomes a major bottleneck in enzyme discovery that has traditionally focused on easilycultivated organisms which are now known to represent less than 1% of biodiversity. Phase I research and development milestones were met or exceeded. In particular, we successfully demonstrated a highefficiency sequencing workflow that will allow us to sequence and assemble our clone library, which is predicted to encode ~600M genes, >99% of which are derived from uncultivated and essentially unstudied organisms. We next demonstrated a combinatorial barcoding strategy that yields assemblies with an average length of >30 kilobases, a dramatic improvement in metagenomic contiguity. This feature enables the discovery of clusters of functionally related genes, such as those that encode complex natural products and nutrient fixation. These services were successfully integrated into an online search engine and ecommerce platform available at www.eve.bio. Finally, we developed and demonstrated infrastructure for an automated LIMS gene recovery system that can recover thousands of genes of interest from our arrayed library per week. The success of Phase I research was complemented by general improvements in sequencing costefficiency and cloudcomputing. The EVE service has gained commercial traction and we believe further development will benefit basic research while positively impacting a broad range of biomanufacturing processes. Based on customer feedback, the aims of this proposal are 1) continued sequencing of the library using contiguitypreserving strategies 2) scaling of computational infrastructure 3) development of advanced enzyme selectors and 4) thirdparty database integration. The overall outcome of this program will be a centralized search engine which allows endusers to rapidly select and receive genes identified in bioinformatic analyses. These genes will be accessible for lower cost, in less time, and from a greater pool of genetic diversity than existing services. Overall, we believe that our platform will improve our understanding of sequencetofunction relationships and annotation for metagenomic environments, helping to bridge the gap between in silico and biochemical characterization from unexplored pools of genetic diversity.